Apparatus, system, and method for purifying nucleic acids

ABSTRACT

Methods and devices for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter are provided. In one embodiment, the method of the invention comprises passing the mixture through a glass frit under conditions effective to separate the nucleic acids from the extraneous matter. In a more specific embodiment, the glass frit is a sintered glass frit.

1 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

This invention relates to the purification of chemical substances, and,more particularly, to devices, methods, and systems for performingchemical purification and analysis. More particularly, the devices,methods, and systems provided by the invention have particularly usefulapplication in the purification and analysis of nucleic acids, and, moreparticularly to microfluidic devices for performing such purificationand analysis. The invention has applications in the areas of analyticalchemistry, forensic chemistry, microfluidics and microscale devices,medicine, and public health.

1.2 The Related Art

The extension of semiconductor fabrication techniques to create highlyminiaturized chemical devices (Beach, Strittmatter et al. 2007) hascreated a revolution in analytical chemistry, especially by providing ameans for identifying chemical substances present in minuteconcentrations in complex mixtures with great precision and accuracy.This revolution has had noticeable impact in chemical processing,medicine, forensic science, and national defense, where such devicesprovide fast, portable, and economic biological detectors. Examples ofsuch devices include devices for collecting and identifying particulates(Wick 2007), systems for detecting molecular contaminants (Knollenberg,Rodier et al. 2007), and devices for detecting proteins (Terry, Scudderet al. 2004; Deshmukh 2006). Other devices use fluidic technologies toisolate and/or amplify nucleic acids using Polymerase Chain Reaction(PCR) in an automated system. Examples of such devices are those soldcommercially by Qiagen (Hilden, Germany), Roche (Basel, Switzerland),Applied Biosystems (Foster City, Calif.), Idaho Technologies (Salt LakeCity, Utah), and Cepheid (Sunnyvale, Calif.).

But as with any analytical process, preparing the sample prior toprocessing is critical to good performance. The presence of too manycomplicating factors and concentrations of substances that may maskanalytes of interest can render robust detection all but impossible.This problem is of particular concern when attempting to analyze thenucleic acid content of cell lysates, which are extremely complex andheterogenous mixtures (Colpan 2001). The preparatory task is made stillmore difficult where portable analytical devices are concerned, sincethose devices are expected to be used in locations where commonlaboratory support equipment, such as centrifuges and separationcolumns, are not available. In those cases, some means for filtering araw sample, such as a blood or urine sample, is critical to providingmeaningful results. Current devices based on fluidic technologies, inparticular the above-mentioned Qiagen devices, use glass filters thatare soft and compliant, requiring a support matrix. The filters havesmall pore sires, typically between about one- and three microns, to getefficient capture of the nucleic acids from the sample. Because of thesmall pores sires, the filters are also relatively thin, typically lessthan two millimeters thick to reduce fluid flow resistance when sampleis forced though the small pore sires. In the Qiagen procedure,typically a sample is mixed with a chaotropic agent, such as guanidine,and the mixture is passed through the glass filter using centrifugalforce, in which fluid flows in only one direction. Nucleic acids bind tothe glass filter; they are washed with ethanol or isopropanol, andsubsequently released using a ten millimolar (10 mM) Tris buffer at a pHof about eight (pH 8.0) or water. But the small pore sizes limit theamount of sample that can be processed, due to resistance created byfluid flow and potential for clogging created by greater flow rates.Thus, devices such as the Qiagen devices can be easily damaged orotherwise rendered ineffective easily. Moreover, these characteristicslimit sample input volume, the types of samples that can be examined,large-volume samples, concentration factors, and simple fluidicintegration.

Larger glass filters have been used to provide pre-processing filtrationof samples. For example, U.S. Pat. No. 4,912,034 (Kalra, Pawlak et al.1990) describes an immunoassay for detecting a target analyte in aliquid sample that includes an optional prefilter assembly made of glassfibers. However, this device is not a microfluidic device and does notshow or suggest the use of glass frits as a filter prior to microscalePCR reactions. U.S. Pat. No. 4,923,978 (McCormack 1990) describes prioruses of glass fiber filters to remove unwanted protein- and protein-DNAcomplexes from aqueous DNA samples, but in a disparaging manner notingthat such filters have low binding capacities (see Column 2). Indeed,the '978 patent claims a very different material for performing suchfiltrations. U.S. Pat. No. 6,274,371 (Colpan 2001) describes silica gel,aluminum oxide, and diatomaceous earth as a preferred filtering agentfor removing unwanted contaminants from cellular lysates prior tonucleic acid analysis. U.S. Pat. No. 6,800,752 (Tittgen 2004) describesusing a chromatography material to separate mixtures comprising nucleicacids, in which the material includes carrier and ion exchangerfunctions wherein the carrier comprises a fibrous material on a support,such as a plastic frit.

Nevertheless, there remains therefore a need to provide fluidic devicesthat are effective to isolate and identify nucleic acids that overcomethe limitations of the current generation of such devices. The presentinvention meets these and other needs.

2 SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for isolatingnucleic acids from a mixture containing such nucleic acids andextraneous matter. Suitable nucleic acids for use in the presentinvention include microbial DNA and human genomic DNA. In oneembodiment, the method of the invention comprises passing the mixturethrough a glass frit under conditions effective to separate the nucleicacids from the extraneous matter. In a more specific embodiment, theglass frit is a sintered glass frit. In some embodiments, the glass frithas a pore size between about 2 microns and about 220 microns; in morespecific embodiments, the glass frit has a pore size between about 150microns and about 200 microns; in other more specific embodiments, theglass frit has a pore size between about 2 microns and about 100microns; and still more specifically, the glass frit has a pore sizebetween about 40 microns and about 75 microns; yet other more specificembodiments includes those in which the glass frit has a pore sizebetween about 2 microns and about 20 microns. In another embodiment, themethod of the invention includes passing the mixture through a glassfrit to produce thereby a first-filtered mixture and then passing thefirst-filtered mixture through a second glass frit under conditionseffective to separate the nucleic acids from the first-filtered mixture.

In a second aspect, the present invention provides a device forfiltering isolating nucleic acids from a mixture containing such nucleicacids and extraneous matter. In some embodiments, the device comprises ahollow chamber having an inlet and an outlet; and disposed therein atleast one glass frit having a pore size of between about 2 microns andabout 220 microns and arranged at a location intermediate the inlet andthe outlet. In more specific embodiments, the glass frit is a sinteredglass frit. In other embodiments, the glass frit is a sintered glassfrit. In some embodiments, the glass frit has a pore size between about2 microns and about 220 microns; in more specific embodiments, the glassfrit has a pore size between about 150 microns and about 200 microns; inother more specific embodiments, the glass frit has a pore size betweenabout 2 microns and about 100 microns; and still more specifically, theglass frit has a pore size between about 40 microns and about 75microns; yet other more specific embodiments includes those in which theglass frit has a pore size between about 2 microns and about 20 microns.

In a third aspect, the present invention provides a fluidic device foridentifying one or more nucleic acids from a mixture of such nucleicacids and extraneous matter. In some embodiments, the fluid device ofthe invention comprises: an inlet, an outlet, and at least one fluidicreaction chamber intermediate the inlet and the outlet and incommunication with each of the inlet and the outlet. The device furthercomprise at least one glass frit arranged at a location (or locations)proximal to the inlet and the reaction chamber(s) and in fluidiccommunication with each of the inlet and reaction chamber(s). The glassfrit(s) have a pore size of between about 2 microns and about 220microns. The mixture enters the device through the inlet and passesthrough the glass frit to exit therefrom as a filtered product beforeentering the fluidic reaction chamber(s). At least one fluidic reagentdispenser is arranged intermediate the glass frit and the reactionchamber(s) and in fluidic communication therewith. In a more specificembodiment, the glass frit is a sintered glass frit. In someembodiments, the glass frit has a pore size between about 2 microns andabout 220 microns; in more specific embodiments, the glass frit has apore size between about 150 microns and about 200 microns; in other morespecific embodiments, the glass frit has a pore size between about 2microns and about 100 microns; and still more specifically, the glassfrit has a pore size between about 40 microns and about 75 microns; yetother more specific embodiments includes those in which the glass frithas a pore size between about 2 microns and about 20 microns. In anotherembodiment, the fluidic device includes a heater proximal to the glassfrit(s).

These and other aspects and advantages will become apparent when theDescription below is read in conjunction with the accompanying Drawings.

3 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a glass frit device (“a filtermodule”) for purifying nucleic acids in accordance with the presentinvention.

FIGS. 2A and 2B are illustrations of a filter in accordance with thepresent invention. FIG. 2A is an illustration an exploded view of aglass frit device (“a filter module”) for purifying nucleic acids inaccordance with the present invention. FIG. 2B is a cut-awayillustration of the same device.

FIG. 3 is a schematic illustration of a glass frit device (“a hollowchamber”) for purifying nucleic acids in accordance with the presentinvention.

FIG. 4 is a schematic illustration of a microarray device in accordancewith the present invention.

FIG. 5 is a flowchart of a process for purifying and identifying nucleicacids in accordance with the present invention.

FIG. 6 is graph illustrating the improved performance characteristics ofdevices in accordance with the present invention as shown in FIGS. 2Aand 2B as demonstrated by measuring fluorescence of a sample material(100 μL of Bacillus anthracis cells in whole blood at a concentration of1×10⁵ cells/mL) as a function of PCR cycles. The solid trace (A) showsthe result for samples treated in accordance with the present invention;the dashed trace (B) shows sample treated using a device availablecommercially from Qiagen; dashed traces (C) and (D) are unprocessedsample and negative control.

FIG. 7 is graph illustrating the improved performance characteristics ofdevices in accordance with the present invention as shown in FIG. 3 asdemonstrated by measuring fluorescence of a sample material (500 μL ofBacillus anthracis cells in sputum at a concentration of 1×10⁴ cells/mL)as a function of PCR cycles. The solid trace (A) shows the result forsamples treated in accordance with the present invention; the dashedtrace (B) shows unprocessed sample; dashed trace (C) is negativecontrol.

4 DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The present invention provides methods and devices for at leastpartially purifying nucleic acids from mixtures of such in combinationwith other substances, such as proteins, small molecules, cell membranefragments, and the like.

As used herein, “nucleic acids” refers to individual nucleic acids andpolymeric chains of nucleic acids, including DNA and RNA, whethernaturally occurring or artificially synthesized (including analogsthereof), or modifications thereof, especially those modifications knowto occur in nature, having any length. Examples of nucleic acid lengthsthat are in accord with the present invention include, withoutlimitation, lengths suitable for PCR products (e.g., about 50 base pairs(bp)) and human genomic DNA (e.g., on an order from about kilobase pairs(Kb) to gigabase pairs (Gb)). Thus, it will be appreciated that the term“nucleic acid” encompasses single nucleic acids as well as stretches ofnucleotides, nucleosides, natural or artificial, and combinationsthereof, in small fragments, e.g., expressed sequence tags or geneticfragments, as well as larger chains as exemplified by genomic materialincluding individual genes and even whole chromosomes. In more specificembodiments, the nucleic acids are from a pathogen, such as bacteria ora virus. Such pathogens include those harmful to humans and animals. Ofthe former, in some embodiments, the pathogen is one that used as abiological weapon, including naturally occurring pathogens that havebeen weaponized. In some embodiments, the nucleic acids comprisemicrobial DNA. In one embodiment of the invention, the microbial DNA isfrom Bacillus anthracis. In other embodiments, the nucleic acids comefrom humans or animals. In some embodiments, the nucleic acids comprisehuman genomic DNA.

In a first aspect, the present invention provides methods for isolatingnucleic acids from a mixture containing such nucleic acids andextraneous matter. In some embodiments, the methods of the inventioncomprise passing the mixture through a hollow chamber having an inletand an outlet; wherein said inlet and outlet are the same and disposedtherein at least one porous filter, under conditions effective toseparate substantially the nucleic acids from the extraneous matter. Asused herein “extraneous matter” refers to all materials that aredistinct from the nucleic acids in the sample. Examples of suchextraneous materials include, but are not limited to, proteins,starches, lipids, metal ions, and larger cellular structures such asmembrane fragments. The phrase “separate substantially” as used hereinrefers to separations that, in some embodiments, provide the nucleicacids in at least 30% purity with respect to the extraneous materials,in more specific embodiments provide the nucleic acids in at least 50%purity with respect to the extraneous materials, in still more specificembodiments provide the nucleic acids in at least 70% purity withrespect to the extraneous materials, in yet more specific embodimentsprovide the nucleic acids in at least 95% purity with respect to theextraneous materials, and in still yet more specific embodiments,provide the nucleic acids in at least 99% purity with respect to theextraneous materials.

In the various embodiments of the invention described herein, the glassfrit is made from standard materials using standard methods as known topersons having ordinary skill in the art or available commercially asdescribed below. In some embodiments, the glass frit has a thicknesssubstantially between about one millimeter and about 20 millimeters,more specifically between about two millimeters and about fivemillimeters, and still more specifically between about two millimetersand about three millimeters. Exemplary glass frit pore sizes suitablefor use with the present invention, including the various embodimentsdescribed herein, are between about 2 microns and about 200 microns. Inmore specific embodiments, the pore size is between about 150 micronsand about 200 microns. In other more specific embodiments, the pore sizeis between about 2 microns and about 100 microns, and still morespecifically between about 40 microns and about 75 microns. Otherembodiments include those for which the pore size is between about 2microns and about 20 microns. For applications involving microbial DNA,a glass frit size of between about 10 microns and about 15 microns issuitable. Larger frit pore sizes can be used for human genomicapplications. Suitable glass frits are composed of sintered glass andare typically used in chemistry glassware and are available commerciallyfrom Robu (Germany). The choice and manufacture of such glass frits willbe understood by persons having ordinary skill in the art.

In other embodiments, the glass frit is replaced or used in conjunctionwith a porous filter. As used herein, “porous filter” refers to anymaterial that allows selective passage of at least one substancecontained in a liquid. More specifically, “porous filter” refers tothose materials capable of substantially removing nucleic acids from aliquid containing such nucleic acids. Examples of suitable porousfilters include, but are not limited to, filter papers configured totrap nucleic acids (e.g., FTA paper, available from Whatman), glassfibers, glass beads, beads with the Charge Switch Technology coating,available from Invitrogen, aluminum oxide filters and porous monolithicpolymers. Such materials and products are familiar to those havingordinary skill in the art. In some embodiments, the porous filter is aglass frit. In further embodiment the glass frit is a sintered glassfrit. Other suitable materials for providing the filtering function ofthe glass frit or sintered glass frit are silicone and XTRABIND (Xtrana,Inc., Broomfield, Colo.). The configuration of such materials to performthe filtering functions of the present invention will be apparent topersons having ordinary skill in the art.

In one embodiment, the above-described glass frit is packaged into afrit holder or fluidic module. One exemplary embodiment of such a holderor module is shown in cut-away view at 1000 in FIG. 1. There, a glassfrit as just described above (1004) in placed inside a housing (1008).The housing includes an inlet (1012) into which fluidic mixtureincluding nucleic acids of interest enter the housing and interact withthe glass frit as described herein to produce a first-filtered mixturewhich passes through an outlet of the housing (1016). After exiting thefilter holder or module, the first-filtered mixture can proceed to otherchambers in fluidic contact with the outlet, such as described below, orto a collector. An optional heater, shown at 1020, is included in someembodiments. The design and manufacture of such devices will beunderstood by those having ordinary skill in the art.

A second embodiment of this aspect of the invention is shown in FIGS. 2Aand 2B. The design and fabrication of such devices are known to thosehaving ordinary skill in the art. FIG. 2A shows an exploded view of oneembodiment of a frit holder (2000), which includes an upper housing body(2002) and a lower housing body (2004). The lower housing body includesa recess (2006), described in greater detail in FIG. 2B, into which isdisposed one or more glass frits (2008) which are described above. Theglass frit is seated in the housing bodies using gaskets (2010, 2012).The lower housing body (2004) also includes an inlet (not shown) throughwhich materials containing nucleic acids to be separated are introducedto glass frit, and outlet (2014) from which waste material and thepurified nucleic acids exit the filter module. FIG. 2B shows a cut-awayview of the frit housing (2000). There, in addition to the elements justdescribed, the inlet (2018) is shown, along with channels for directingthe flow of material through the glass frit and outlet.

In another aspect, the present invention provides a device for isolatingnucleic acids from a mixture containing such nucleic acids andextraneous matter. One embodiment of such a filter in accordance withthe present invention is shown at 3000 in FIG. 3. There, a hollowchamber (3004) having a first opening (3008) and a second opening (3012)through which a mixture comprising nucleic acids is passed and an outletfrom which an at least partially resolved mixture exits. Between theinlet and outlet is a glass frit (3018) as described above that extendsaxially through at least a portion of the interior volume of the hollowchamber, the extent to which is shown at 3016. In some embodiments, morethan one such frit is used. In still other embodiments, at least one ofthe glass frits is made of sintered glass. The design and fabrication ofsuch devices are known to those having ordinary skill in the art.

In some more specific embodiments, one end of the hollow chamber has afrustaconical shape the chamber is dimensioned to fit on the end of apipetting instrument, e.g., as a pipet tip, so that materials are firsttaken up though the second opening, pass through the glass frit, arefiltered and then retained in the portion of the chamber above the frit.In some embodiments, the sample retained in the portion of the chamberabove the frit is passed back through the frit through the secondopening (3012).

In another embodiment, the above-described pipet tip is combined with aheating device that is configured to heat the frit to facilitateseparation of the nucleic acids from the mixture. In more specificembodiments, the heater is dimensioned to fit within the pipet tip. Thedesign and fabrication of such devices are known to those havingordinary skill in the art.

In still another embodiment, the pipet tip is coupled with an electronicpipettor or robotic pipetting workstation to control the flow ratethrough the frit. In some embodiments, the electronic pipettor is ahand-held device. The design, fabrication, and operation of such devicesare known to those having ordinary skill in the art.

In some embodiments of the present invention, two or more porous filtersare used in combination. In a more specific embodiment, each layer has adifferent pore size. Without wishing to be bound to any particulartheory of action, larger porous filter pore sizes trap larger particles,and so can serve as a prefilter. For example, a 40 micron-60 micronporous filter could be used in tandem with a 10 micron-15 micron porousfilter to deplete human genomic DNA from a sample (e.g., blood) toisolate microbial DNA. Removing the abundant human DNA with the 40micron-60 micron porous filter allows better binding of the low copymicrobial DNA to the 10 micron-15 micron porous filter and more robustanalysis, since the human genomic DNA will not be present inconcentrations large enough to interfere significantly (e.g., by wholegenome amplification). The porous filter can be in a pipet tip asdescribed above, having a thickness and diameter of about fivemillimeters (mm) each. In some embodiments, two or more of the porousfilters are fused together to form a substantially monolithic structure.The design, fabrication, and operation of such devices are known tothose having ordinary skill in the art.

In more specific embodiments in which the filter is disposed with apipet tip, the glass frit(s) having larger pore sizes are disposedcloser to the pipet tip inlet. Again not wishing to be bound to anyparticular theory of action, but those persons having ordinary skill inthe art will appreciate that arranging the larger pore sized filternearer the pipet tip inlet can provide a more uniform distribution ofnucleic acid binding within the frit. By way of comparison, personshaving ordinary skill in the art will expect that otherwise the nucleicacids will tend to bind to the frit at the area closest to the pipet tipopening, since the nucleic acids are more likely to make initial contactjust as the nucleic acids enter the frit.

In yet another aspect, the present invention provides a microfluidicdevice for analyzing nucleic acids in accordance with the presentinvention. One embodiment of such a microfluidic device is show in FIG.4 at 4000. A frit holder (4002) as described herein is provided.Upstream, the frit holder in fluidic communication with a source ofelution buffer (4004), a guanidine hydrochloride (Gu) reservoir (4006)and Gu mixing tower (4008), the flow from which Gu and Gu mixing towerare controlled by a valve (4010). The Gu mixing tower is further influidic communication with an ethanol-air source (4012). Those personshaving ordinary skill in the art will realize that chaotropes other thanGu can be used with the present invention. In addition are a bead beater(4014) that is in fluidic communication with a sample collection tower(4016), which is in turn in fluidic communication with inlet check value(4018), and an electrical contact (4020). The output from these elementsis controlled by valve 4022.

With continuing reference to FIG. 4, downstream of frit holder (4002) isa waste tank (4024), the flow to which is controlled by a valve (4026).Downstream flow from the frit holder is also controlled by a secondvalve (4028), which controls flow to an elution tower (4030) and a checkvalve (4032) along a first branch; and to another valve (4034) along asecond branch of the flow path. Continuing downstream from valve 4034,are one or more reservoirs of PCR reagents (4036) and a valve (4038)which leads to a PCR chamber (4040). Downstream of the PCR chamber is avalve (4042), which, along with valve 4046, controls flow from the PCRchamber to a microarray chamber (4048) that is also in fluidiccommunication with hybridization and wash buffer reservoir (4048) and awaste container (4052). The design and fabrication of such devices areknown to those having ordinary skill in the art.

The operation of the device described with respect to FIG. 4 isillustrated by the flowchart shown in FIG. 5 at 5000. After obtainingthe raw sample (5002), e.g., a sputum sample that contains cells ofinterest, the cells are lysed (5004) using bead beater 4014, and themixture passed for purification of the nucleic acids (5006) through thefrit 4002 for amplification (5008) by PCR chamber 4040 and detection(5010) by the microarray chamber 4050.

Without being bound to any particular theory or action, the presentinvention meets the needs described above by implementing a rigid,self-supporting frit structure that is relatively thick for high bindingcapacity, contains relatively large porosities for low fluid impedance,faster flow rates, and higher tolerance to particles in clinical andenvironmental samples, and consists of no loose material (e.g. silicagel, diatomaceous earth, glass beads) and no flimsy, delicate materials(e.g. fiber filters, membrane filters, silicon microstructures) forrugged operation and packaging and simplified manufacturing.

5 EXAMPLES

The following Examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in the art inpracticing the invention. These Examples are in no way to be consideredto limit the scope of the invention in any manner.

5.1 Protocol for Using a Device of the Invention

Referring to FIGS. 2A and 2B, a protocol for practicing purification anddetection in accordance with the present invention is provided below.

-   -   1. Insert a glass frit into holder (one of four different        porosities: Fine, Medium. Coarse, and Extra Coarse). Tighten the        housing.    -   2. Mix 500 μL of a sample (10⁴ copies/mL) with 500 μL of 6M        guanidine, pH 6.5.    -   3. Pass mixture (1 mL) through frit at a flow rate of 100 μL/min        using a 1 mL syringe. Pass air manually through frit to purge        sample using a 5 mL syringe.    -   4. Pass 1 mL of 70% ethanol (EtOH) to wash bound nucleic acid        using a 1 mL syringe at rate of 1 mL/min. Pass air through the        frit manually to purge EtOH using a 5 mL syringe.    -   5. Carefully pass an elution buffer (10 mM Tris, pH 8.0) using 1        mL syringe at 100 μl/min into the frit holder until buffer can        be first seen in the outlet tubing.    -   6. Place heat block under the frit holder and heat at 70° C. for        3 min.    -   7. After 3 minutes continue to pass elution buffer through frit        holder. Collect the fractions (50 μL-100 μL) for PCR analysis.    -   8. Flush the frit holder with 1 mL of 10% bleach (bleach        dilution no more than one week old), 5 mL of 10 mM Tris-HCl (pH        8.0), and 5 mL of water. Replace the frit.

5.2 Second Protocol for Practicing the Invention

Referring to FIG. 3, a protocol for practicing purification anddetection in accordance with the present invention is provided below.

-   -   1. Add 500 μL of sample to 500 μL of 6M guanidine in Vial A.        Vortex to mix.    -   2. Attach a 1.2 mL pipet tip with an embedded frit to an        electronic pipettor (Gilson Concept).    -   3. Set electronic pipettor to speed 1 (slowest speed). Aspirate        the 1 mL of sample mixture in Vial A. Allow the sample mixture        bolus to completely pass through the frit. The sample mixture        bolus will establish itself immediately on top of the frit.    -   4. Dispense the sample back into Vial A. The sample mixture        bolus will completely expel back into the vial.    -   5. Repeat steps 3 and 4 four times.    -   6. Set electronic pipettor to speed 5 (fastest speed). Aspirate        and dispense 1 mL of 70% ethanol in Vial B to wash bound nucleic        acids on frit. Repeat four times.    -   7. Remove traces of ethanol by positioning tip above the ethanol        solution. Aspirate and dispense air five times to dry the frit.    -   8. Place Vial C containing 100 μL of 10 mM Tris-HCl (pH 8.0)        into a heat block set at 70° C. Let heat for 5 minutes.    -   9. Set the electronic pipettor to speed 1. Aspirate the elution        buffer and dispense back into Vial C five times to remove        nucleic acids from the frit.

5.3 Demonstration of Superior Results from the Invention

The results of two experiment using such protocols are show in FIGS. 6and 7, where samples of Bacillus anthracis (Ba) in whole blood andsputum were treated using the materials and methods of the invention.

In FIG. 6, the sample treated using a method and device (FIGS. 2A and2B) of the invention (trace A) outperformed a sample treated using adevice available commercially from Qiagen (trace B). Traces C and D arethe unprocessed input sample and negative control, respectively. In theexperiment, 100 μL of Bacillus anthracis cells at 10⁵/mL in whole bloodwas input, either through a medium (10-15 μm pore size) frit asdescribed herein or into a Qiagen device, and elutions of about 50 μLwere collected and analyzed using PCR. As the Figure illustrates, afterabout 25 cycles of PCR amplification, the sample treated using thematerials and methods of the invention clearly and significantlyoutperform the results from the sample treated using the prior artdevice.

FIG. 7 illustrates the result of another experiment using such a methodand device (FIG. 3), where a sample of Bacillus anthracis cells insputum was treated using the materials and methods of the invention. Thesample treated using the method and device of the invention (trace A)outperformed an unprocessed sample (trace B). Trace C is a negativecontrol. In the experiment, 500 μL of Bacillus anthracis cells at 10⁴/mLin sputum was input through a medium frit as described herein, and anelution of about 100 μL was collected and analyzed using quantitative,real-time PCR. As the Figure illustrates, after about 27 cycles of PCRamplification, the sample treated using the material and method of theinvention clearly and significantly outperform the results fromunprocessed sample.

The above-described methods and devices were used successfully toidentify DNA and/or RNA from a variety of organisms listed in Table 1that were spiked into a variety of matrices (i.e., sample types) listedin Table 2.

TABLE 1 Viral Equine Encephalitis (VEE) Vaccinia virus Y. pestis B.anthracis Adenovirus S. pyogenes C. pneumoniae Influenza A Influenza BMixture of Adenovirus + S. pyogenes Mixture of Flu A and Adenovirus

The following materials were analyzed successfully using the methods anddevices of the invention:

TABLE 2 Water/TE (10 mM Tris-HCl, 1.0 mM EDTA buffer) Swab extractsSputum Nasal Wash Whole Blood

6 CONCLUSION

The present invention offers several advantages over the prior art: 1) asimplified method and device requiring no centrifugation or highpressure to move fluids, 2) a modular device for fluidic integrationinto a complete analytical system, 3) large pore sizes to processcomplex samples while maintaining low fluid resistance; 4) highextraction and elution efficiencies since fluids can be moved from bothdirections; 5) rigidity that eliminates the need for additional supportstructures, and 6) durability that allows reusing the device forsequential samples. Although various specific embodiments and exampleshave been described herein, those having ordinary skill in the art willunderstand that many different implementations of the invention can beachieved without departing from the spirit or scope of this disclosure.For example, other materials with nucleic acid affinity could be usedfor the glass frit or the glass frit can be modified to better attractnucleic acids and nucleic acids without using a chaotropic salt such asguanidine. Glass frits can contain immobilized antibodies to extractmicrobes and toxins. Still other variations will be clear to thosehaving ordinary skill in the art.

7 BIBLIOGRAPHY

The following references are incorporated herein by reference in theirentirety and for all purposes.

-   Beach, R. A., Strittmatter, R. P., et al (2007). Integrated    Micropump Analysis Chip and Method of Making the Same U.S. Pat. No.    7,189,358.-   Colpan, M. (2001). Process and device for the Isolation of Cell    Components, Such as Nucleic Acids, from Natural Sources U.S. Pat.    No. 6,274,371.-   Deshmukh, A. J. (2006). Method and Automated Fluidic System for    Detecting Protein in Biological Sample U.S. Pat. No. 6,989,130.-   Kalra, K. L., Pawlak, K., et al. (1990). Immunoassay Test Device and    Method U.S. Pat. No. 4,912,034.-   Knollenberg, B. A., Rodier, D., et al. (2007). Molecular    Contamination Monitoring System and Method U.S. Pat. No. 7,208,123.-   McCormack, R. M. (1990). Process for Purifying Nucleic Acids U.S.    Pat. No. 4,923,978.-   Terry, B. R., Scudder, K. M., et al. (2004). Method and Apparatus    for High Density Format Screening for Bioactive Molecules. U.S. Pat.    No. 6,790,652.-   Tittgen, J. (2004). Chromatography Material and a Method Using the    Same U.S. Pat. No. 6,800,752.-   Wick, C. H. (2007). Method and System for Detecting and Recording    Submicron Sized Particles. U.S. Pat. No. 7,250,138.

1. A device for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter, comprising: a hollow chamber having disposed therein at least one glass frit that is capable of binding to said nucleic acids in a cell lysate prepared by lysing cells outside said glass frit, said glass frit being arranged within said hollow chamber such that said mixture flows through said glass frit when said mixture is flowed through said hollow chamber, and said glass frit having a pore size of between about 2 microns and about 20 microns, wherein said glass frit comprises a rigid, self-supporting frit structure with a thickness of about 1-20 mm and wherein said glass frit is not modified with a material with nucleic acid affinity.
 2. The device of claim 1, wherein said glass frit is a sintered glass frit.
 3. The device of claim 2, further comprising a heating device configured to heat said glass frit.
 4. The device of claim 1, wherein said glass frit has a pore size between about 10 microns and about 15 microns.
 5. A fluidic device for identifying one or more nucleic acids from a mixture of such nucleic acids and extraneous matter, comprising: an inlet, an outlet, and at least one fluidic reaction chamber intermediate said inlet and said outlet and in communication with each of said inlet and said outlet; said device further comprising at least one glass fit arranged at a location proximal to said inlet and said at least one reaction chamber and in fluidic communication with each of said inlet and at least one fluidic reaction chamber, said glass frit having a pore size of between about 2 microns and about 20 microns and being capable of binding to said one or more nucleic acids in a cell lysate prepared by lysing cells outside said glass frit, such that said mixture enters said device through said inlet and passes through said glass frit to exit therefrom as a filtered product before entering said at least one fluidic reaction chamber; at least one fluidic reagent dispenser arranged intermediate said glass frit and said at least one reaction chamber, said at least one fluidic reagent dispenser being in fluidic communication with said at least one fluidic reaction chamber, wherein said glass frit comprises a rigid, self-supporting frit structure with a thickness of about 1-20 mm and wherein said glass frit is not modified with a material with nucleic acid affinity.
 6. The device of claim 5, wherein said at least one glass frit includes at least one sintered glass frit.
 7. The device of claim 5, wherein said at least one glass frit includes at least one glass frit having a pore size between about 10 microns and about 15 microns.
 8. The device of claim 5, further comprising a heater proximal to at least one said at least one glass frit. 